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Down-regulation of CYLD expression by Snail promotes tumor progression in malignant melanoma.

Massoumi R, Kuphal S, Hellerbrand C, Haas B, Wild P, Spruss T, Pfeifer A, Fässler R, Bosserhoff AK - J. Exp. Med. (2009)

Bottom Line: Rescue of CYLD expression in melanoma cells reduced proliferation and invasion in vitro and tumor growth and metastasis in vivo.Importantly, tumor thickness and progression-free and overall survival inversely correlated with CYLD expression.Our data suggest that Snail1-mediated suppression of CYLD plays a key role in melanoma malignancy.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Medicine, Max Planck Institute of Biochemistry, 82152 Martinsried, Germany.

ABSTRACT
High malignancy and early metastasis are hallmarks of melanoma. Here, we report that the transcription factor Snail1 inhibits expression of the tumor suppressor CYLD in melanoma. As a direct consequence of CYLD repression, the protooncogene BCL-3 translocates into the nucleus and activates Cyclin D1 and N-cadherin promoters, resulting in proliferation and invasion of melanoma cells. Rescue of CYLD expression in melanoma cells reduced proliferation and invasion in vitro and tumor growth and metastasis in vivo. Analysis of a tissue microarray with primary melanomas from patients revealed an inverse correlation of Snail1 induction and loss of CYLD expression. Importantly, tumor thickness and progression-free and overall survival inversely correlated with CYLD expression. Our data suggest that Snail1-mediated suppression of CYLD plays a key role in melanoma malignancy.

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CYLD regulates proliferation and N-cadherin–mediated migration and invasion. (A) Proliferation of Mel Im cells expressing CYLD, CYLD C/S, or GFP versus the parental Mel Im cell line (Control) after 72 h. Data are given as mean ± SEM. *, P < 0.05. (B) Colony formation in soft agar 3 wk after plating 104 Mel Im cells expressing CYLD, CYLD C/S, or GFP versus the parental (Control) cell line. (C) Migration of melanoma cells in monolayer scratch assays. Mel Im cells transduced with lentiviral vectors carrying CYLD, CYLD C/S, or GFP and noninfected control cells (Control) were used. Representative pictures (at day 0–2) and calculation (mean ± SEM; *, P < 0.05) of the areas between scratch fronts (after 2 d). (D) Migration of Mel Im cells (Control) compared with CYLD, CYLD C/S, or GFP stably transduced cells in spheroid migration assays. Data are given as mean ± SEM. *, P < 0.05. (E) Invasion of Mel Im (Control) cells compared with CYLD, CYLD C/S, or GFP stably transduced cells (after 24 h) in Boyden chamber assays. Data are given as mean ± SEM. *, P < 0.05 compared with Control and GFP. (F) Migration of melanoma cells in Boyden chamber assays. Comparison of Mel Im cells stably transduced with CYLD, CYLD C/S or GFP, and transiently cotransfected with an N-cadherin expression vector (+N-cadherin) or control vector (+pCMX). Data are given as mean ± SEM. *, P < 0.05. (G) Migration of melanoma cells in monolayer scratch assays. Comparison of nontransduced Mel Im cells (Control), cells transfected with BCL-3 siRNA nucleotides (siRNA BCL-3), or scrambled siRNA control (siRNA Control). Data are given as mean ± SEM. *, P < 0.05 compared with Control or siRNA control. Experiments in A–G were repeated at least three times.
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fig5: CYLD regulates proliferation and N-cadherin–mediated migration and invasion. (A) Proliferation of Mel Im cells expressing CYLD, CYLD C/S, or GFP versus the parental Mel Im cell line (Control) after 72 h. Data are given as mean ± SEM. *, P < 0.05. (B) Colony formation in soft agar 3 wk after plating 104 Mel Im cells expressing CYLD, CYLD C/S, or GFP versus the parental (Control) cell line. (C) Migration of melanoma cells in monolayer scratch assays. Mel Im cells transduced with lentiviral vectors carrying CYLD, CYLD C/S, or GFP and noninfected control cells (Control) were used. Representative pictures (at day 0–2) and calculation (mean ± SEM; *, P < 0.05) of the areas between scratch fronts (after 2 d). (D) Migration of Mel Im cells (Control) compared with CYLD, CYLD C/S, or GFP stably transduced cells in spheroid migration assays. Data are given as mean ± SEM. *, P < 0.05. (E) Invasion of Mel Im (Control) cells compared with CYLD, CYLD C/S, or GFP stably transduced cells (after 24 h) in Boyden chamber assays. Data are given as mean ± SEM. *, P < 0.05 compared with Control and GFP. (F) Migration of melanoma cells in Boyden chamber assays. Comparison of Mel Im cells stably transduced with CYLD, CYLD C/S or GFP, and transiently cotransfected with an N-cadherin expression vector (+N-cadherin) or control vector (+pCMX). Data are given as mean ± SEM. *, P < 0.05. (G) Migration of melanoma cells in monolayer scratch assays. Comparison of nontransduced Mel Im cells (Control), cells transfected with BCL-3 siRNA nucleotides (siRNA BCL-3), or scrambled siRNA control (siRNA Control). Data are given as mean ± SEM. *, P < 0.05 compared with Control or siRNA control. Experiments in A–G were repeated at least three times.

Mentions: To investigate the functional role of the suppressive effect of CYLD on Cyclin D1 and N-cadherin, we used the two different melanoma cell lines (Mel Im and Mel Juso) stably expressing CYLD (Fig. 4 A). CYLD expression caused diminished proliferation in Mel Im cells (Fig. 5 A) and Mel Juso cells (Fig. S5 A, available at http://www.jem.org/cgi/content/full/jem.20082044/DC1), as well as a decrease in colony growth in soft agar in both cell lines (Fig. 5 B and Fig. S5 B) compared with control, GFP, or CYLD C/S–infected cells.


Down-regulation of CYLD expression by Snail promotes tumor progression in malignant melanoma.

Massoumi R, Kuphal S, Hellerbrand C, Haas B, Wild P, Spruss T, Pfeifer A, Fässler R, Bosserhoff AK - J. Exp. Med. (2009)

CYLD regulates proliferation and N-cadherin–mediated migration and invasion. (A) Proliferation of Mel Im cells expressing CYLD, CYLD C/S, or GFP versus the parental Mel Im cell line (Control) after 72 h. Data are given as mean ± SEM. *, P < 0.05. (B) Colony formation in soft agar 3 wk after plating 104 Mel Im cells expressing CYLD, CYLD C/S, or GFP versus the parental (Control) cell line. (C) Migration of melanoma cells in monolayer scratch assays. Mel Im cells transduced with lentiviral vectors carrying CYLD, CYLD C/S, or GFP and noninfected control cells (Control) were used. Representative pictures (at day 0–2) and calculation (mean ± SEM; *, P < 0.05) of the areas between scratch fronts (after 2 d). (D) Migration of Mel Im cells (Control) compared with CYLD, CYLD C/S, or GFP stably transduced cells in spheroid migration assays. Data are given as mean ± SEM. *, P < 0.05. (E) Invasion of Mel Im (Control) cells compared with CYLD, CYLD C/S, or GFP stably transduced cells (after 24 h) in Boyden chamber assays. Data are given as mean ± SEM. *, P < 0.05 compared with Control and GFP. (F) Migration of melanoma cells in Boyden chamber assays. Comparison of Mel Im cells stably transduced with CYLD, CYLD C/S or GFP, and transiently cotransfected with an N-cadherin expression vector (+N-cadherin) or control vector (+pCMX). Data are given as mean ± SEM. *, P < 0.05. (G) Migration of melanoma cells in monolayer scratch assays. Comparison of nontransduced Mel Im cells (Control), cells transfected with BCL-3 siRNA nucleotides (siRNA BCL-3), or scrambled siRNA control (siRNA Control). Data are given as mean ± SEM. *, P < 0.05 compared with Control or siRNA control. Experiments in A–G were repeated at least three times.
© Copyright Policy
Related In: Results  -  Collection

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fig5: CYLD regulates proliferation and N-cadherin–mediated migration and invasion. (A) Proliferation of Mel Im cells expressing CYLD, CYLD C/S, or GFP versus the parental Mel Im cell line (Control) after 72 h. Data are given as mean ± SEM. *, P < 0.05. (B) Colony formation in soft agar 3 wk after plating 104 Mel Im cells expressing CYLD, CYLD C/S, or GFP versus the parental (Control) cell line. (C) Migration of melanoma cells in monolayer scratch assays. Mel Im cells transduced with lentiviral vectors carrying CYLD, CYLD C/S, or GFP and noninfected control cells (Control) were used. Representative pictures (at day 0–2) and calculation (mean ± SEM; *, P < 0.05) of the areas between scratch fronts (after 2 d). (D) Migration of Mel Im cells (Control) compared with CYLD, CYLD C/S, or GFP stably transduced cells in spheroid migration assays. Data are given as mean ± SEM. *, P < 0.05. (E) Invasion of Mel Im (Control) cells compared with CYLD, CYLD C/S, or GFP stably transduced cells (after 24 h) in Boyden chamber assays. Data are given as mean ± SEM. *, P < 0.05 compared with Control and GFP. (F) Migration of melanoma cells in Boyden chamber assays. Comparison of Mel Im cells stably transduced with CYLD, CYLD C/S or GFP, and transiently cotransfected with an N-cadherin expression vector (+N-cadherin) or control vector (+pCMX). Data are given as mean ± SEM. *, P < 0.05. (G) Migration of melanoma cells in monolayer scratch assays. Comparison of nontransduced Mel Im cells (Control), cells transfected with BCL-3 siRNA nucleotides (siRNA BCL-3), or scrambled siRNA control (siRNA Control). Data are given as mean ± SEM. *, P < 0.05 compared with Control or siRNA control. Experiments in A–G were repeated at least three times.
Mentions: To investigate the functional role of the suppressive effect of CYLD on Cyclin D1 and N-cadherin, we used the two different melanoma cell lines (Mel Im and Mel Juso) stably expressing CYLD (Fig. 4 A). CYLD expression caused diminished proliferation in Mel Im cells (Fig. 5 A) and Mel Juso cells (Fig. S5 A, available at http://www.jem.org/cgi/content/full/jem.20082044/DC1), as well as a decrease in colony growth in soft agar in both cell lines (Fig. 5 B and Fig. S5 B) compared with control, GFP, or CYLD C/S–infected cells.

Bottom Line: Rescue of CYLD expression in melanoma cells reduced proliferation and invasion in vitro and tumor growth and metastasis in vivo.Importantly, tumor thickness and progression-free and overall survival inversely correlated with CYLD expression.Our data suggest that Snail1-mediated suppression of CYLD plays a key role in melanoma malignancy.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Medicine, Max Planck Institute of Biochemistry, 82152 Martinsried, Germany.

ABSTRACT
High malignancy and early metastasis are hallmarks of melanoma. Here, we report that the transcription factor Snail1 inhibits expression of the tumor suppressor CYLD in melanoma. As a direct consequence of CYLD repression, the protooncogene BCL-3 translocates into the nucleus and activates Cyclin D1 and N-cadherin promoters, resulting in proliferation and invasion of melanoma cells. Rescue of CYLD expression in melanoma cells reduced proliferation and invasion in vitro and tumor growth and metastasis in vivo. Analysis of a tissue microarray with primary melanomas from patients revealed an inverse correlation of Snail1 induction and loss of CYLD expression. Importantly, tumor thickness and progression-free and overall survival inversely correlated with CYLD expression. Our data suggest that Snail1-mediated suppression of CYLD plays a key role in melanoma malignancy.

Show MeSH
Related in: MedlinePlus